Prior work by us and others has demonstrated that the extracellular pH (pHe) of solid tumors is commonly, and perhaps always, acidic. This proposal seeks to continue our research to better understand the role of acid pHe in the evolutionary dynamics of cancer progression and therapy. We have observed that an acidic pHe is important, and perhaps necessary, for the transition from an in situ to an invasive cancer. By facilitating invasion, an acidic pHe is also a critical factor in the formation of metastases. This grant is unique within the NCI portfolio by focusing on tumor pH, which is a fundamental property of cancer. Over its 14-year history, it has been organized around three interrelated themes to: 1) improve measurement of pHe; 2) understand the causes of acidosis; and 3) exploit the consequences of acid pHe for therapeutic benefit. This organization has been very productive and there are compelling new challenges in each of these themes that will be addressed in the next period of support. Over the last 14 years of work in Aim 1, we and others have developed increasingly sophisticated methods to measure the pHe in animal tumor models. With the advent of novel acid pH targeted therapies, however, there is a compelling need for methods to measure pH in human patients. We therefore propose to predict the acidic habitat using a combination of MRI and PET imaging. Because there will be some limitations to this approach, we will continue to develop hyperpolarized HCO3 as a pH-imaging agent, because there is a clinical trajectory of hyperpolarized MRI. Over the course of Aim 2, we have quantitatively identified that metabolism-perfusion mismatch is a key driver of tumor acidity. However, during the last period of support, it has become increasingly understood that there is a great amount of intra- tumoral heterogeneity, not only in habitats and genomics, but also in metabolic profiles that are sure to have an impact on intratumoral pH. We thus propose to develop a quantitative understanding of this through immunohistochemistry and metabolic profiling of cells and complex tissues. This work will not only contribute to our basic understanding of cancer biology, it will also identify and test novel therapeutic targets. Studies f Aim 3 have defined effects of pH on chemotherapy, and have led to the discovery that neutralization of tumor acidity with buffers also inhibited metastasis. Over the last period of support, we have continued to investigate buffer therapy and have initiated clinical trials. There are still many unanswered questions with this approach, and this will be the focus during the next period of support. The proposed work will focus exclusively on pancreatic ductal adenocarcinoma, PDAC, for a number of compelling reasons. First among these is that alternative therapies are desperately needed for this cancer and, second, preliminary data have demonstrated regional acidosis plays a critical role in the biology and clinical course of PDAC. We expect this work will result in biomarker-driven clinical trials for buffer therapy combinations in PDAC patients.